Contaminant removal method

ABSTRACT

A method for purifying Apo A-I is provided including the steps of providing a solution comprising Apo A-I and guanidine hydrochloride and filtering the solution through a filter having a pore size in a range from 15 nm to 35 nm to thereby reduce viral contamination of the Apo A-I. An Apo A-I preparation is provided having at least a 12 log LRV (log reduction value) for a parvovirus; and/or at least 9 log LRV for a non-enveloped virus; and/or at least 8.5 log LRV for a lipid enveloped virus. Also provided are pharmaceutical compositions and reconstituted high density lipoprotein formulation comprising Apo A-I and methods of treating diseases disorders or conditions.

TECHNICAL FIELD

The invention relates to a method for purifying apolipoprotein, inparticular for removing viral pathogens from apolipoprotein A-I (ApoA-I) containing solutions and to provide an Apo A-I preparation.

BACKGROUND

Apolipoproteins are the major protein component in soluble lipoproteincomplexes with apolipoprotein A-I (Apo A-I) being the major proteincomponent in high density lipoprotein (HDL) particles.

The apolipoproteins of the A, C and E families have evolved from acommon ancestral gene and are structurally similar. These proteinmolecules generally contain a series of 22-amino acid tandem repeatsthat are often separated by proline residues. The repeating 22-aminoacid segments form amphipathic α-helices which enable binding to bothlipid and water surfaces. In the case of human Apo A-I (243 amino acids;28.1 kDa) there are eight 22-mer and two 11-mer amphipathic helices(Lund-Katz & Phillips, 2010, Subcell Biochem. 51, 183-227). Theamphipathic α-helices of the apolipoproteins play a critical role instabilising the lipoprotein. They do this by orientating theapolipoprotein so that the predominantly hydrophobic helical faces caninteract with the hydrophobic lipids in the complex whilst the opposingpredominantly hydrophilic faces of the apolipoprotein interact with thesurrounding aqueous environment. However when these proteins areseparated from the lipid component, exposure of the hydrophobic aminoacid residues to an aqueous environment can make them difficult tohandle. In particular the hydrophobic faces of the α-helices have atendency to self-associate resulting in aggregate formation and in someconditions precipitation. For example, a 1 mg/mL solution containing ApoA-I Milano is estimated to contain 80% of the protein in an aggregatedform when stored in 50 mM phosphate buffer at pH 7A (Suurkuust & Hallen,2002, Spectroscopy 16, 199-206).

Apo A-I is synthesized by the liver and intestine and is responsible forthe physiological function of HDL in the blood; the removal ofcholesterol from peripheral tissues, carrying it back either to theliver or to other lipoproteins, by a mechanism known as “reversecholesterol transport” (RCT). As a consequence the HDL particles arepresent in plasma in a range of sizes and are continually remodellingdue to these RCT lipid transfer activities. Thus HDL particles arecharacterized by a high density (>1.063 g/ml) and sizes ranging fromabout 5 to 20 nm (Stoke's diameter). The clear correlation betweenelevated levels of serum cholesterol and the development of coronaryheart disease (CHD) has been repeatedly confirmed, based onepidemiological and longitudinal studies. Hence, Apo A-I in HDL isthought to have an anti-inflammatory function and to restrain theoccurrence and development of CHD. Furthermore, Apo A-I has shown todecrease the Low Density Lipoproteins (LDL) level in the blood and isknown to bind to lipopolysaccharides or endotoxins, thus having a majorrole in the anti-endotoxin function of HDL. The “protective” role of HDLand Apo A-I as the primary protein constituent has been confirmed in anumber of studies. High plasma levels of Apo A-I are associated with areduced risk of CHD and presence of coronary lesions. Apo A-I is thuspromising for applications in drugs like reconstituted HDL forapplications in acute coronary syndromes, atherosclerosis treatment,anti-inflammation treatment, antitoxin treatment, liver-targeting drugs,etc.

Biological therapeutics of either recombinant or plasma origin arecommonly manufactured using biological feed-stocks that areintrinsically contaminated with pathogens such as viruses. Moreover,some manufacturing processes are, by their nature, susceptible topathogen contamination from extrinsic sources. Accordingly,manufacturers of biological therapeutics are required to incorporatesufficient virus clearance steps into their manufacturing processes toensure that their products are contaminant-free.

Biotechnology products (typically proteins or DNA) are produced withrecombinant DNA in cell cultures, transgenic animals, or transgenicplants. Common cells used for production include Chinese Hamster Ovary(CHO) cells, E. coli bacteria, and yeast. Cell-based production systemsare typically carried out in batch mode although a small number ofperfusion systems are also in use. Final commercial scale fermentationis carried out at 1,000-100,000 L scale with the majority of CHO basedfermenters in the 8,000-25,000 L scale.

Human blood plasma is nowadays collected in large amounts (for example,it has been estimated that in 2010 that 30 million litres of plasma werecollected worldwide) and processed to individual fractions; some ofthese fractions contain the apolipoprotein, Apo A-I. Examples of suchplasma fractions include Cohn Supernatant I, Cohn Fraction II+III, andCohn Fraction IV (e.g. Cohn Fraction IV-1) or variations thereof (e.g.is a Kistler/Nitschmann Fraction IV). Since blood and plasma potentiallycontain transfusion-transmissible pathogens, such pathogens, inparticular viruses must be removed or inactivated when blood- orplasma-derived components are used as therapeutics or as a vehicle fortherapeutic delivery. However, viruses are often not easily removed andmay still be present in plasma-derived components, even if they arehighly purified. In particular, small non-enveloped viruses such asPicornaviridae (e.g. hepatitis A virus) which have a size of about 27-32nm and Parvoviruses which have a size of about 18-26 nm, are of specialconcern. This is due to both their small size and their highphysiochemical stability. Thus, there is an ongoing need for thedevelopment of methods that allow for efficient virus removal orinactivation of plasma-derived protein therapeutics.

Common virus inactivation technologies include physical methods such asthe classical pasteurization procedure (60° C. heating for 10 hours),short wavelength ultra-violet light irradiation, or gamma irradiationand chemical methods such as solvent detergent or low pH incubation.Virus removal technologies include size exclusion methods such as virusfiltration which is also often referred to as nanofiltration. Thesevirus filtration methods have been shown to be effective methods forremoving viruses from protein solutions.

Virus filtration has the benefit of being a mild method for removingviruses from protein solutions, and generally allows for a high level ofprotein recovery and the biological activity of the proteins to be fullypreserved. Optimal virus filters must maximize capacity, throughput, andselectivity. The capacity of a virus filter is the total volume offiltrate per m² of filter surface area that can be processed before theflux declines to an unacceptably low value during constant pressurefiltration. Throughput refers to the speed at which the feed can befiltered (maximum sustainable permeate flux). Selectivity refers to theability to yield high product recovery and high virus particleretention. These filters must be able to process the entire bulk feed atacceptable filtrate fluxes, reject virus particles, and maximize proteinpassage. Fouling during virus filtration is typically dominated byprotein aggregates, DNA, partially denatured product, or other debris.

Filter manufacturers often assign terms like nominal or mean pore sizeratings to commercial filters, which usually indicate meeting certainretention criteria for particles or microorganisms rather than thegeometrical size of the actual pores.

For viral clearance, filtration is conducted through a filter membrane,which has a nominal pore size smaller than the effective diameter of thevirus which is to be removed. The presence of only a small number ofabnormally large pores (300 kDa or larger nominal molecular weightcutoff, NMWCO) will permit excessive virus leakage. Hence virus filtersmust be manufactured so as to eliminate all macro-defects. This istypically accomplished through the use of composite membranes thatprovide the required combination of virus retention and mechanicalstability. Virus-removing filter membranes are typically made frommaterials such as regenerated cellulose, for example acuprammonium-regenerated cellulose or synthetic polymer materials likehydrophilic polyvinylidene fluoride (PVDF) or hydrophilicpolyether-sulfone (PES) as described in the literature: Manabe. S,Removal of virus through novel membrane filtration method., Dev. Biol.Stand., (1996) 88: 81-90.; Brandwein H et al., Membrane filtration forvirus removal., Dev Biol (Basel)., (2000) 102: 157-63.: Aranha-Creado etal., Clearance of murine leukaemia virus from monoclonal antibodysolution by a hydrophilic PVDF microporous membrane filter.,Biologicals. (1998) June; 26 (2): 167-72.; Mocé-Llivina et al.,Comparison of polyvinylidene fluoride and polyether sulfone membranes infiltering viral suspensions, Journal of Virological Methods, (2003)April, Vol. 109, Issue 1, Pages 99-101.

Virus filtration methods that have been described include WO96/00237which relates to a method of virus-filtering a solution that containsmacromolecules (i.e. protein) by adding salt to the solution to a levelof at least 0.2 M. The applicants recommend using salts that exhibit thehigh salting-out effect that is characteristic of the high end of theHofmeister series, in particular citrate, tartrate, sulfate, acetate orphosphate anions and sodium, potassium, ammonium or calcium cations.Sodium chloride is particularly preferred, and salts that exhibit a lowsalting-out effect at the low end of the series (e.g. GuHCl) are notused.

Consistent with WO96/00237, Kim et al (Biotechnology & BioprocessEngineering, 2011, 16, 785-792) describe the virus filtration of Apo A-Iin the presence of sodium chloride (250 mM NaCl, 30 mM Tris at pH 8).This step is carried out immediately after elution from a DEAE-FFcolumn. The propensity for Apo A-I to aggregate however imposes amanufacturing limitation in that the filtration step ideally needs to becompleted either as the Apo A-I is eluted from the column oralternatively the Apo A-I needs to be stored in the presence of salt atvery low protein concentrations (e.g. 0.1 mg/mL). This later approachhas the disadvantage of then requiring overly large filtration volumes.The cost of virus filters is substantial. Thus any reduction in filtercapacity due to for example apolipoprotein aggregation or overly largefiltration volumes can result in significantly higher processing costsat commercial scale.

WO03/105989 relates to the use of clathyrate modifiers such as polyolsugar or sugar alcohol (i.e. sucrose and sorbitol) and is aimed atincreasing the hydrophobicity of the filter membrane surface anddecreasing the hydrodynamic radius of the protein as well as reducingthe tendency for the self-association of the protein desired to befiltered.

US 2003/232969 A1 relates to a method for removing viruses fromsolutions of high molecular weight proteins like fibrinogen (340 kDa) bynanofiltration.

Apolipoproteins like Apo A-I being relatively small (28 kDa) should bereadily amenable to virus filtration. However as already described abovetheir hydrophobic nature along with their unfortunate tendency to formaggregates promote the formation of protein clusters on the filtersurface and also to clogging the pores of the filter. In terms of thefilter itself, this can occur on the upper surface of the membrane, bothby pore blockage and/or by the formation of a cake or deposit, and alsowithin the membrane pore structure. Fouling causes decay in flow ratefor constant pressure operation and increases the pressure for operationat constant filtrate flux. As a result of filter fouling there can bereductions to the selectivity of the filtration resulting in lowerprotein recoveries and/or lower virus retention.

Additionally filter fouling reduces the capacity and throughputresulting in longer filtration times and/or the requirement forincreased filter area. In addition, operating conditions which areoptimal for maintaining apolipoprotein solubility and preventingaggregation might not be optimal for ensuring a high viral clearance. Inparticular chaotrophic substances that might be used to stabilise theapolipoprotein may alter the filterability properties of the pathogen(e.g. virus) and possibly also the filter membrane. As a consequence,the presence of a chaotrophic substance at particular concentrationsmight also allow unwanted virus penetration across the filter membrane,thus negating the usefulness of the step for processing solutionscomprising apolipoproteins.

It is therefore an object of the present invention to provide afiltration method for safely removing viruses, in particular smallnon-enveloped viruses such as parvoviridae, which is suitable forsolutions comprising apolipoproteins, like Apo A-I, and which is alsosuitable for industrial application.

SUMMARY

This object is broadly achieved by a method for purifying apolipoproteinA-I (Apo A-I), that comprises the filtering a solution comprising ApoA-I and guanidine hydrochloride (GuHCl) through a filter having asuitable pore size.

According to an aspect of the present invention, there is provided amethod for purifying apolipoprotein A-I (Apo A-I), comprising the stepsof: a) providing a solution comprising Apo A-I and guanidinehydrochloride (GuHCl); and b) filtering the solution through a filterhaving a pore size in a range from 15 nm to 35 nm.

In an embodiment, the method is for reducing viral contamination of theApo A-I. In some embodiments of the invention, the method is forreducing viral contamination of the Apo A-I wherein the viralcontamination comprises a parvo virus. In particular embodiments theparvo virus is minute virus of mice (MVM). In some embodiments of theinvention, the method is for reducing viral contamination of the Apo A-Iwherein the viral contamination comprises a picomaviridae virus. Inparticular embodiments the picomaviridae virus is encephalomyocarditis(EMCV) or hepatitis A.

In a preferred embodiment, the solution comprises an Apo A-I proteinconcentration within a range from 5 to 30 g/L, particularly from 5 to 20g/L, for example 7 to 12 g/L.

In a preferred embodiment, the solution comprises a GuHCl concentrationthat reduces or inhibits aggregation of the Apo A-I. The solution may inparticular comprise a GuHCl concentration within a range from 1.3 to 3.2M, particularly from 1.5 to 2.0 M. More preferably the GuHClconcentration in the solution is 1.7 M.

In an embodiment, the pH of the solution is within a range from 7.1 to7.5, such as at 7.3.

In an embodiment, the solution is prepared by one or more steps of: 1)suspending Apo A-I precipitate in 4.0 to 4.6 M GuHCl; and/or 2) dilutingthe suspension to an Apo A-I protein concentration within a range from 5to 30 g/L, and/or to a GuHCl concentration within a range from 1.3 M to3.2 M.

In an embodiment, after step b), a heat treatment step is performed forvirus inactivation. The heat treatment may comprise the steps of:adjusting the pH of the solution within a range from 6.6 to 8.0; andsubsequently heating the solution at a temperature of 55 to 61° C. forabout 30 minutes to about 4 hours.

In an alternative embodiment, prior to step a), a heat treatment step isperformed for virus inactivation. The heat treatment may comprise thesteps of: providing a solution comprising GuHCl and Apo A-I at a pHwithin a range from 6.6 to 8.0; and subsequently heating the solution ata temperature of 55 to 61° C. for about 30 minutes to about 4 hours.

In preferred embodiments the solution with a pH within the range from6.6 to 8.0 comprises a GuHCl concentration within a range from 2.7 to3.9 M, or more preferably 3.5 M.

In embodiments of the invention, the pH of the solution with a pH withinthe range from 6.6 to 8.0 is preferably within a range from 7.0 to 8.0.In particular embodiments the pH is 7.3.

An aspect of the present invention also provides Apo A-I purified by themethod of the invention.

An aspect of the present invention also provides an Apo A-I preparationwith at least 12 log LRV (log reduction value) for a parvovirus; and/orat least 9 log LRV for a non-enveloped virus; and/or at least 8.5 logLRV for a lipid enveloped virus. In particular embodiments the Apo A-Ipreparation is suitable for pharmaceutical use.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising Apo A-I or the Apo-AI preparation according tothe aforementioned aspects together with a pharmaceutically acceptablecarrier, diluent or excipient.

In yet another aspect, the invention provides a method of producing apharmaceutical composition including producing Apo A-I according to themethod of the aforementioned aspect and combining the Apo A-I with apharmaceutically acceptable carrier or diluent; or combining the Apo A-Ipreparation of the aforementioned aspect with a pharmaceuticallyacceptable carrier or diluent; to thereby produce the pharmaceuticalcomposition.

A further aspect of the invention provides an rHDL formulationcomprising Apo A-I or the Apo-AI preparation according to theaforementioned aspects together with a lipid.

In another further aspect, the invention provides a method of producinga reconstituted HDL formulation including producing Apo A-I according tothe method of the aforementioned aspect and combining the Apo A-1 with alipid; or combining the Apo A-I preparation of the aforementioned aspectwith a lipid; to thereby produce the reconstituted HDL formulation.

A yet further aspect of the invention provides a method of treating orpreventing a disease, disorder or condition in a mammal including thestep of administering to the mammal Apo-AI, an Apo-A1 preparation, apharmaceutical composition or a rHDL formulation according to any of theaforementioned aspects to a mammal to thereby treat or prevent thedisease, disorder or condition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: showing through-put of the solution [kg/m²] duringnanofiltration with conditions of 3.2 M GuHCl, 19.7 g/L Protein.

FIG. 2: showing through-put of the solution [kg/m²] duringnanofiltration with conditions of 1.7 M GuHCl, 8.9 g/L Protein.

FIG. 3: showing through-put of the solution [kg/m²] duringnanofiltration with conditions of 1.7 M GuHCl, 5.8 g/L Protein.

FIG. 4: showing the load and flux throughout the nanofiltration.

DETAILED DESCRIPTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

The reference in this specification to any prior publication (orinformation derived from it), or to any matter which is known, is not,and should not be taken as an acknowledgment or admission or any form ofsuggestion that that prior publication (or information derived from it)or known matter forms part of the common general knowledge in the fieldof endeavour to which this specification relates.

It must be noted that, as used in the subject specification, thesingular forms “a”, “an” and “the” include plural aspects unless thecontext clearly dictates otherwise. Thus, for example, reference to “aprotein” includes a single protein, as well as two or more proteins.

The term “about” in relation to a numerical value x means, for example,x±10%.

Where the invention provides a process involving multiple sequentialsteps, the invention can also provide a process involving less than thetotal number of steps. The different steps can be performed at verydifferent times by different people in different places (e.g. indifferent countries).

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

The term “pore size” in the context of a filter typically means the sizeof pores in the filter. Typically, the pore size is smaller or less thanthe size of the smallest viruses that can be removed by the filter. Inthis context, while certain embodiments of the invention may refer togeometric properties of pore size (e.g. diameter), it will beappreciated that pore size can be functionally defined, as will bedescribed in more detail hereinafter.

It has surprisingly been found that when using guanidine hydrochloride(GuHCl) (sometimes also referred to as guanidine chloride or abbreviatedas GdHCl, GdmCl or GndCl) according to the present invention, an Apo A-Isolution can be filtered through a filter having pores with a pore sizein the 15 nm to 35 nm range to achieve substantial or complete viralclearance, yet without aggregation of the proteins and clogging thefilters. In particular embodiments the filter has a pore size in a rangefrom 15 nm to 35 nm; or from 15 nm to less than 35 nm; or from 15 nm to30 nm; or from 15 nm to 25 nm; or from 15 nm to 20 nm; or from 20 nm to25 nm; or from 18 nm to 23 nm; or from 15 nm to 26 nm; or from 18 nm to26 nm; or from 27 nm to 32 nm; or from 25 nm to 30 nm; or from 20 nm to30 nm.

In embodiments of the invention, a filter having a pore size in a rangefrom 15 nm to 35 nm has a mean pore size in the range from 15 nm to 35nm. In some embodiments the filter has a mean pore size in the rangefrom 15 nm to less than 35 nm; or from 15 nm to 30 nm; or from 15 nm to26 nm; or from 18 nm to 26 nm; or from 15 nm to 25 nm; or from 15 to 20nm; or from 20 nm to 25 nm; or from 18 nm to 23 nm; or from 27 nm to 32nm; or from 25 nm to 30 nm; or from 20 nm to 30 nm. In particularembodiments the mean pore size is about 15 nm; or about 20 nm; or about25 nm; or about 30 nm; or about 35 nm.

The use of GuHCl is beneficial from two points of view: 1) due to itschaotropic properties, GuHCl functions as an anti-aggregation agent andthus inhibits the formation of aggregate clusters of for example the ApoA-I proteins in solution; 2) however, it does not irreversibly affectthe apolipoprotein structure so that once the GuHCl is removed, Apo A-Ihelices reform and the protein is able to associate with lipids to formparticles, like reconstituted HDL. Moreover the apolipoprotein maintainsits biological activity, which is highly important for the further useof the product as a pharmaceutical substance.

An advantage of the present invention is that it is possible to increasethe rate of flow and to decrease the liquid volumes as well as thefilter areas and process time needed for the filtration. As a result,larger amounts of the Apo A-I solution can be filtered within a shorttime, increasing the efficiency of the process substantially.

Viruses that can be removed efficiently with the present invention canhave a size smaller than about 300 nm. The size of the viruses that canbe removed suitably is smaller than about 200 nm. Examples of suchviruses include cytomegalovirus (about 180-200 nm, plasma products);herpes simplex virus (about 150-200 nm, recombinant products); andepstein-barr virus (about 120-200 nm, recombinant antibody products).The size of the viruses that can be removed is preferably smaller thanabout 120 nm. Examples of such viruses include HIV (about 80-120 nm,plasma derived products). Normally, the viruses that can be removed arelarger than about 20 nm, i.e. the approximate size of the parvo virus(15-26 nm, plasma and recombinant derived products). The parvo viruses,like B19 (plasma products) at about 18-26 nm in size are difficult toremove from a therapeutic protein source, so nanofiltration is typicallyconsidered a critical manufacturing step to evaluate for viral clearanceas the filter pore sizes in the 20 nm range are close to the known sizeof the parvoviruses. Nevertheless, the process of the present inventionallows for safe and efficient removal of small viruses such as membersof the parvovirus family and hepatitis A (about 27-32 nm, plasmaproducts), hepatitis B (about 42 nm, plasma products), hepatitis C(about 30-60 nm, plasma products) and encephalomyocarditis (about 25-30nm; recombinant products) viruses. This is particularly noteworthy,since according to the state of the art, excessive amounts liquids fordilution are required in order to prevent aggregation of the proteinsand clogging of the filters, which often resulted in break-through ofsmall viruses.

In particular embodiments of the invention the filter has a pore sizecapable of removing from a solution comprising Apo A-I and guanidinehydrochloride (GuHCl) a parvovirus such as MVM or B19; and/or ahepatitis A virus; and/or a picomaviridae virus such asencephalomyocarditis virus.

Virus retention is suitably characterized in terms of the Log ReductionValue (LRV), which is defined as the logarithm (base 10) of the ratio ofthe viral concentration in the feed to that in the filtrate: LRV=−log10S; where S is the sieving coefficient for the virus. The totalrequired LRV depends on the nature and potential for viral contaminationof the starting material. Virus filtration steps are typically designedto provide a minimum of 4-log virus removal (LRV). Viral clearancestudies are performed by spiking high titer infectious viruses (withdifferent physical characteristics) into scaled-down modules andevaluating the LRV. Removal of both enveloped and non-enveloped virusesmay be demonstrated. Common model viruses include animal parvoviruses(e.g., MVM, B19), poliovirus (non-specific model virus),encephalomyocarditis virus (EMCV) (model for picomaviridae viruses likehepatitis A), simian virus 40 (SV40, non-specific model virus), sindbisvirus (model for hepatitis C), bovine viral diarrhoea virus (BVDV, modelfor flaviviridae viruses like hepatitis C), duck hepatitis B virus(model for hepatitis B), Japanese Encephalitis virus (model forhepatitis C) and reovirus (non-specific model virus). Initial designstudies can also be performed with bacteriophages which can be obtainedat much higher purity and titers, and which are much easier (and lessexpensive) to assay.

Virus filtration validation studies are usually performed at small scaletypically using self-contained devices with 13-47 mm discs. All processparameters are scaled down in a linear fashion and should representworst-case conditions with regard to virus clearance. It is alsoimportant to process a larger amount of the feed stream per surface areacompared with the industrial scale process design to insure validationunder worst case process conditions. Virus clearance should be measuredin several fractions. Several recent studies have demonstrated thatvirus clearance can decrease with the extent of membrane fouling(reduction in permeability) when using parvo-type virus filters (20 nmpore size).

The method of the present invention provides a very efficient way forinactivating and/or removing viruses. In principle, supposing that suchhigh viral content is actually present, the present invention allows forthe reduction of the content of very small non-enveloped viruses, suchas the parvovirus by at least 3 log LRV (lowering the number of virusesby 1,000-fold), suitably by at least 4 log LRV (10,000-fold reduction),suitably by at least 5 log LRV (100,000-fold reduction) and preferablyby at least 6 log LRV (1,000,000-fold reduction in the number ofviruses).

In addition, the filtration method according to the invention can beeasily combined with other virus removing or inactivating procedures,such as a heat treatment step, for ensuring even higher viral depletion.

Optionally, a pre-filtration or clarifying filtration step can beperformed before the virus filtration in order to remove macro-sizeparticles. Such a pre-filtration can also be performed with a filtercomprising a membrane with a larger pore diameter than that of thevirus-removing membrane. In an embodiment of the invention thepre-filter has a pore size in the range of 0.05-0.5 μm. In particularembodiments the pre-filters are selected from Pall Nylon membrane filter(SKL 7002 NTP 0.1 μm or FTKNI) or the Sartopore 2 filter. In particularembodiments the pre-filter size is chosen by using at least 0.025 m² offilter surface area per kg of starting material Apo A-I precipitate. Inother embodiments the pre-filter size is chosen by using at least 0.014m² of filter surface area per kg of starting material Apo A-Iprecipitate. In particular embodiments the pre-filter size is in therange from about 0.014 m² to about 0.035 m² of filter surface area perkg of starting material Apo A-I precipitate. The pre-filtration can beconducted either in line with the virus filter or out of line withrespect to the virus filter. In particular embodiments thepre-filtration is conducted in line with respect to the virus filter. Inparticular embodiments the pre-filter is made from the same membranematerial as the virus removing filter.

Suitable filters for the virus filtration method according to theinvention are available commercially and can be purchased, for example,under the designations such as Planova BioEx (Asahi Kasei Corporation),inter alia. Such filters are sometimes referred to as ‘small virus’removal filters.

In particular embodiments the virus removing filter comprises a membranemanufactured of one or more materials selected from cuprammoniumregenerated cellulose, hydrophilic polyvinylidene fluoride (PVDF),composite PVDF, surface modified PVDF, nylon, and polyether sulfone.

In embodiments of the invention the filter membrane is a flat sheet or ahollow fibre membrane. Examples of flat sheet membranes includehydrophilised PVDF filter membranes such as the Pegasus™ Grade SV4small-virus removal filters (Pall Corporation). In an embodiment thefilter comprises a PVDF flat sheet membrane. In an embodiment the filtercomprises a hydrophilic PVDF flat sheet membrane, or a composite PVDFflat sheet membrane, or a surface modified PVDF flat sheet membrane. Ina particular embodiment the filter is the Pegasus™ Grade SV4.

In other embodiments the filter is a hollow fibre membrane. The hollowfibre membrane format typically contains a bundle of straw-shaped hollowfibres with the wall of each hollow fibre containing a 3 dimensional webstructure of pores comprised of voids interconnected by finecapillaries. Examples of hollow fibre filters include the Planova™ BioEXfilters (Asahi Kasei Corporation) which incorporates hydrophilicmodified polyvinylidene fluoride (PVDF) in hollow fibre membrane format.In an embodiment the filter comprises a PVDF membrane in a hollow fibremembrane format. In an embodiment the filter comprises a hydrophilicPVDF hollow fibre membrane format, or a composite PVDF hollow fibremembrane format, or a surface modified PVDF hollow fibre membraneformat. In particular embodiments the filter is the Planova™ BioEX.

For the purpose of comparing filter membranes that can have quitedifferent structures it is not adequate to examine pore size usingvisual methods like microscopy. Hence reference to pore size, asdescribed herein, describes a structural property of the filter assessedwith functional methods, rather than visual methods. Estimation of thefilter pore size can be made using a functional method. Such methodsinclude bubble point measurements, liquid-liquid porosity, intrusionporosimetry, sieving of macromolecules (e.g. bacteriophages) and/orparticles of defined sizes.

Mean flow bubble point can be measured according to ASTM E1294-89(‘Standard Test Method for Pore Size Characteristics of Membrane FiltersUsing Automated Liquid Porosimeter’). Briefly the method involveswetting the filter with perfluorohexane (e.g. Fluorinert™ FC-72) andthen applying a differential pressure of air to remove the fluid. Thedifferential pressure at which wet flow is equal to one half the dryflow (flow without wetting solvent) is used to calculate the mean flowpore size.

In particular embodiments of the invention the filter has a mean flowbubble point measured with perfluorohexane above 100 psi, or above 120psi.

In other embodiments of the invention the pore size of the filter can beestimated by applying a colloidal gold particle solution to the filter(e.g. AGPTS—Asahi Kasei Corporation, gold particle test system). Priorto the beginning of the test, an in-line visual wavelength spectrometermeasures the initial absorbance. As the gold particle solution passesthrough the filter, a second absorbance reading evaluates the goldparticle removal rate and a pore size distribution result is determinedbased on an LRV calculation of the absorbance values.

The apolipoprotein may be any apolipoprotein which is a functional,biologically active component of a naturally-occurring HDL or of areconstituted high density lipoprotein (rHDL). Particularapolipoproteins include members of the A, C and E families. Typically,the apolipoprotein is either a plasma-derived or recombinantapolipoprotein such as Apo A-I, Apo A-II, Apo A-V, pro-Apo A-I or avariant such as Apo A-I Milano or so called oxidation resistant formssuch as 4WF. In particular embodiments the apolipoprotein is Apo A-I. Insome embodiments the Apo A-I is derived from plasma. In otherembodiments the Apo A-I is recombinant Apo A-I. Preferably, the Apo A-Iis either recombinantly derived comprising a wild type sequence or theMilano sequence or it is purified from human plasma. The Apo A-I can bein the form of monomers, dimers, or trimers, or multimers or mixturesthereof. The apolipoprotein may be in the form of a biologically-activefragment of apolipoprotein. Such fragments may be naturally-occurring,chemically synthetized or recombinant. By way of example only, abiologically-active fragment of Apo A-I preferably has at least 50%,60%, 70%, 80%, 90% or 95% to 100% or even greater than 100% of thelecithin-cholesterol acyltransferase (LCAT) stimulatory activity of ApoA-I.

The starting material containing an apolipoprotein like Apo A-I may bederived, for instance, from Precipitate IV according to Kistler andNitschmann fractionation method, which has been further purified, e.g.by cold ethanol precipitation. In alternative embodiments the startingmaterial containing an apolipoprotein like Apo A-I is a cellculture/fermentation extract. In embodiments the Apo A-I is produced bycell culture in a E. coli host/vector system or a mammalian host cellincluding but not limited to Chinese hamster ovary (e.g., CHO-KI orCHO-S), VERO, BHK, BHK 570, HeLa, COS-I, COS-7, MDCK, 293, 3T3, PC12 andW138, or from an amyeloma cell or cell line (e.g., a murine myeloma cellor cell line). In particular embodiments the cell cultures may becultivated in a serum free medium. In embodiments the cell cultures maybe cultivated in a serum free medium lacking animal derived components.

Before use, Apo A-I precipitate may be stored at a temperature below−20° C. in the freezer.

For suspending the Apo A-I precipitate, the volume ratio of Apo A-Iprecipitate to solution can be in the range from 1:2 to 1:5. In someembodiments the volume ratio of Apo A-I precipitate to solution is inthe range 1:3 to 1:4. In particular embodiments the volume ratio of ApoA-I precipitate to solution is 1:3, or 1:3.1, or 1:3.2, or 1:3.3, or1:3.4, or 1:3.5.

In order to facilitate the re-suspension, the frozen Apo A-I precipitatecan be broken into small pieces (<5 cm of diameter) inside apolyethylene bag by using e.g. a pharma-hammer before adding the frozenApo A-I precipitate to the solution.

The suspension can be subsequently diluted with WFI (water forinjection) for adjusting the Apo A-I protein concentration and the GuHClconcentration in the solution to the desired range.

In some embodiments the solution comprising Apo A-I and guanidinehydrochloride (GuHCl) is derived from a purified Apo A-I as described inPCT/AU2014/000584.

In the context of the present invention, the expression “5 to 30 g/L ApoA-I” and similar expressions mean that 5 to 30 g of apolipoprotein A-I(Apo A-I) protein is solubilized in 1 L solution. The apolipoproteinconcentration for the filtration step is typically in the range of 0.5g/L to 50 g/L. In particular embodiments the Apo A-I proteinconcentration in the solution of step a) is in the range from 5 to 25g/L; or 5 to 20 g/L; or 5 to 15 g/L; or 5 to 12 g/L; or 7 to 12 g/L; or5 to 11 g/L; or 7 to 11 g/L; or 5 to 10 g/L; or 7 to 10 g/L. In someembodiments of the invention the protein concentration of the solutioncomprising Apo A-I and guanidine hydrochloride (GuHCl) is determined bymeasuring the absorbance at 280 nm and then calculating the proteinconcentration as described in Example 1. In some embodiments the proteinconcentration of the solution comprising Apo A-I and guanidinehydrochloride (GuHCl) is determined by nephelometry or high performancecapillary electrophoresis (using the method described in Example 1).

It is particularly preferred that the GuHCl concentration of thesolution of step a) is within the range from 1.3 to 3.2 M. In particularembodiments the GuHCl concentration is within the range from 1.3 to 3.0M; or 1.3 to 2.75 M; or 1.3 to 2.5 M; or 1.5 to 3.0 M; or 1.5 to 2.75 M;or 1.5 to 2.5 M; or 1.5 to 2.25 M; or 1.5 to 2.0 M; or 1.5 to 1.9 M.More preferably the GuHCl concentration is within the range from 1.6 to1.9 M and most preferably the GuHCl concentration is 1.7 M. Thisconcentration is ideal to suppress aggregate formation of the Apo A-Iprotein in the solution, improve the filterability (i.e. capacity andthroughput) and ensure maximum retention of virus in the filter membrane(i.e. selectivity). In embodiments of the invention the concentration ofGuHCl of the solution comprising Apo A-I and guanidine hydrochloride(GuHCl) is determined using ion exchange chromatography. In particularembodiments of the invention the guanidine content is determined usingion chromatography (HPLC) with a suitable cation exchange column (e.g.IonPac CS19 analytical column, 4 ×250 mm (Thermo Scientific, Dionex)).The Dionex IonPac CS19 column can be used with a Reagent-Free™ IonChromatography (RFIC™) system for automatic methanesulfonic acid (MSA)eluent generation and electrolytic eluent suppression with conductivitydetection. In this way the background due to ions from the mobile phase(e.g. methane sulfonic acid, MSA) are suppressed and detection of theguanidine can be carried out by measuring the conductivity. Samplescontaining the Apo A-I and guanidine can be mixed with an internalstandard (e.g. formamidine acetate) and diluted in water so that theguanidine concentration is about 0.1-2.0 mg/mL. The chromatography canbe run in either isocratic or gradient modes. The quantification can bebased on the peak area with a five point calibration (0.1-2.0 mg/mL) andthe internal standard.

According to an embodiment, the pH of the solution before the virusfiltration step is within the range from about 7 to about 10; or fromabout 7 to about 9; or from about 7 to about 8. In particularembodiments the pH of the solution before the virus filtration step iswithin the range from 7.1 to 7.5. In a further embodiment the pH of thesolution before the virus filtration step is at 7.3. This pH range is atphysiological (about 7.35-7.45) or nearly at physiological pH. Ideallythe pH will be at least one pH unit away from the isoelectric point ofthe apolipoprotein. In particular embodiments the pH is at least one pHunit away from the isoelectric point of the apolipoprotein. In the caseof human Apo A-I and variants thereof the isoelectric point is typicallyin the range of about pH 5.2 to 5.8. The isoelectric point of anapolipoprotein can be determined by isoelectric focusing (IEF) such asby the method described in Contiero et. al. (1997) Electrophoresis18(1), 122-126. When multiple apolipoprotein isoform peaks are presentin the IEF profile then the mean isoelectric point can be used.

In the context of the present invention, when the pH of a solution iswithin a given pH range, e.g. a range from 7.1 to 7.5, this means thatthe solution “has” a pH of that range, e.g. a pH of 7.1 to 7.5. Thismeans that the solution is formed at a pH of 7.1 to 7.5, or is after itsformation brought to a pH of 7.1 to 7.5.

In general, the pH is measured either in the solution before adding theApo A-I protein to said solution; or directly after mixing the Apo A-Iprotein with the solution. Typically, the pH of the solution of step a)is measured right after mixing the precursor components. Alternatively,the pH of the mixture can also be determined by calculation based on theprojected amounts and concentrations of the components in the mixture.

In the present invention, solution refers to a solution that contains atleast 50 percent by weight of water, optionally including one or moresolvents, such as methanol or ethanol. The solvents can be anypharmaceutical grade solvent. In particular embodiments of the inventionthe solvent is ethanol for example 95% pharmaceutical grade ethanol(e.g. 3A containing 5% methanol). In a further embodiment the solutioncomprises about 20% ethanol.

Optionally the filter used in step a) is prewashed with WFI and/or GuHClsolution before filtering the solution. This prewashing step increasesthe permeability of the proteins through the filter. In a particularembodiment the filter is prewashed with 1.3 to 2.0 M GuHCl.

In order to minimize the loss of protein through the virus filtration,in some embodiments after the filtration, the filter is post-washed withGuHCl. In particular embodiments the filter is post-washed with 1.3 to2.0 M GuHCl.

The virus filtration can be performed using either tangential flowfiltration (TFF) or ‘dead-end’ filtration (also known as normal ordirect flow filtration). Virus filters were originally designed for usein TFF with the feed flowing adjacent to the upper skin layer of theasymmetric membrane. TFF provides high flux by sweeping the membranesurface to reduce concentration polarization and fouling. However, thesimplicity and lower capital cost of dead end filtration has led to thewidespread use of virus filters specifically designed for dead endfiltration. In contrast to TFF, these dead end filters are typicallyoperated with the more open side of the membrane facing the feed stream,allowing protein aggregates and other large foulants to be capturedwithin the macroporous substructure thereby protecting thevirus-retentive skin layer. Advantages of using single-use dead endfilters include that they simplify both system design and validation,reducing labor and capital costs.

Dead-end filtering typically involves using a single pump to force fluidthrough the membrane from the surface.

Tangential filtration generally requires a first pump to maintainconstant flow rate at the surface of the filter membrane and a secondpump draws the protein through the membrane by creating a negativepressure at the back of the membrane.

In particular embodiments the filtration is performed by dead-endfiltration.

In particular embodiments the dead-end filtration process is conductedusing either constant pressure filtration or constant velocityfiltration. In a particular embodiment the dead-end filtration processis conducted using constant pressure filtration.

Filtration is performed with filtration pressure that is the same as orbelow the level at which the membrane can withstand, depending on thematerial of a virus-removing membrane to be used herein, for examplewith pressures of about 0.2 to about 3.4 bar. In particular embodimentsthe filtration pressure is maintained between about 0.2 bar to about 3.4bar. In embodiments the filtration pressure is maintained at about 1 toabout 3 bar; or at about 1.5 to about 3 bar, or at about 1.7 to about 3bar; or at about 2 to about 3 bar; or at about 2.2 to about 3 bar; or atabout 2.2 to about 2.7 bar. In embodiments the filtration pressure ismaintained at about 1.7 bar to about 2.4 bar; or at about 2.2 bar toabout 2.4 bar.

The temperature has an effect on the viscosity of a protein solution andalso has an effect on the flux upon filtration with a virus-removingmembrane. The solution to be used in the filtration step should have atemperature within the range from 0° C. up to the temperature at whichthe protein concerned is denatured. The temperature of the solutionsuitably is within the range of from about 10° C. up to about 50° C. Inparticular embodiments the temperature of the solution is within therange of from about 18° C. up to about 35° C. In some embodiments thesolution is filtered at room temperature from about 18° C. to about 26°C.

In particular embodiments two or more filters are used in series. In aparticular embodiment the filtration is conducted using two filters inseries having a pore size in a range from 15 nm to 35 nm. In someembodiments the two or more filters have a pore size in the range from15 nm to less than 35 nm; or from 15 nm to 30 nm; or from 15 nm to 25nm; or from 15 to 20 nm; or from 20 nm to 25 nm. In particularembodiments the two or more filters have a mean pore size selected fromthe group of about 15 nm; or about 20 nm; or about 25 nm; or about 30nm; or about 35 nm.

In embodiments of the invention the virus filter capacity is at least200 kg or at least 300 kg or at least 340 kg or at least 500 kg or atleast 750 kg or at least 1000 kg of the solution comprising Apo A-I andGuHCl per m² of filter surface area.

The solution of step a) can be prepared by suspending Apo A-Iprecipitate in a solution comprising 4.0 to 4.6 M GuHCl and subsequentlydiluting the suspension to a desired Apo A-I protein concentrationwithin the range from 5 to 30 g/L and to a desired GuHCl concentrationwithin the range from 1.3 to 3.2 M.

In particular embodiments of the invention the method for purifyingapolipoprotein (Apo A-I) comprises filtering a solution comprising 5 to30 g/L Apo A-1 at a pH from about 7 to about 8 and guanidinehydrochloride (GuHCl) at a concentration of 1.3 to 3.2 M. Wherein, thefilter has a pore size in a range from 15 nm to 35 nm and the filtrationis a dead-end filtration at a pressure of about 1 to about 3 bar and atemperature of about 18° C. to about 35° C.

In particular embodiments of the invention the method for purifyingapolipoprotein (Apo A-I) comprises filtering a solution comprising 5 to20 g/L Apo A-I at a pH from about 7 to about 8 and guanidinehydrochloride (GuHCl) at a concentration of 1.5 to 3.0 M. Wherein, thefilter has a pore size in a range from 15 nm to less than 35 nm and thefiltration is a dead-end filtration at a pressure of about 1 to about 3bar and a temperature of about 18° C. to about 35° C.

In particular embodiments of the invention the method for purifyingapolipoprotein (Apo A-I) comprises filtering a solution comprising 5 to20 g/L Apo A-I at a pH from about 7 to about 8 and guanidinehydrochloride (GuHCl) at a concentration of 1.5 to 2.0 M. Wherein, thefilter has a pore size in a range from 15 nm to 26 nm and the filtrationis a dead-end filtration at a pressure of about 1 to about 3 bar and atemperature of about 18° C. to about 35° C.

As a routine practice, virus filters are integrity tested pre- andpost-use to ensure that the filter achieves the required level ofperformance. To facilitate this, filter manufacturers have developed avariety of destructive and non-destructive physical integrity tests. Thepurposes of these physical integrity tests are to confirm that: (1) thevirus filter is properly installed; (2) the filter is free from defectsand damages; and (3) the performance of the filters is consistent withboth manufacturers' specifications and end-user virus retention studies.The most commonly used nondestructive tests include the bubble pointtest, the forward flow test (e.g. Palltronic Flowstar XC (Pall)), thewater intrusion test and the binary gas test. Both the bubble point testand the forward flow test evaluate a wet membrane as a barrier to thefree flow of a gas. The water intrusion test, also called HydroCorrtest, uses a dry hydrophobic membrane as a barrier to the free flow ofwater, a non-wetting fluid. The binary gas test uses a mixture of twogases with high differences in permeability, and the test is based onmeasurement of the composition of the gas mixture upstream anddownstream of a water-wetted membrane. The gold particle test, thepost-use integrity test used with Planova (Asahi Kasei Corporation)filters, is a destructive integrity test. Generally, nondestructivetests are used due to the option of retesting filter integrity if theinitial test fails. If the post-use integrity tests and retests fail,re-filtration is a common practice for virus filtration steps. Thesetests can additionally be used to estimate filter pore size, asdescribed above.

According to a particular embodiment of the present invention, themethod further comprises a heat treatment step for further viraldepletion. In a preferred embodiment said heat treatment step isconducted prior to step a) (Option I). In an alternative embodiment saidheat treatment step is conducted after step b) (Option II).

The apolipoprotein concentration for the heat inactivation step istypically in the range of 0.5 to 50 g/L. In particular embodiments thatApo A-I protein concentration is within the range from 5 to 30 g/L.

According to Option I of the above embodiment, the heat treatment stepis performed prior to step a), thus before the virus filtration. In thiscase, a solution comprising GuHCl, e.g. in a concentration of 2.7 to 3.9M, and Apo A-I at a pH of 6.6 to 10.0, in particular embodiment at a pHof 6.6 to 8.0, is provided in a first step. The solution is thensubsequently heated at temperature of 55 to 61° C. for about 30 minutesto about 4 hours in order to inactivate viruses that may still bepresent in the solution.

According to Option II of the above embodiment, the heat treatment stepis conducted after step b), thus after the virus filtration. In thiscase, the GuHCl concentration in the solution after step b) is adjustedto provide a solution comprising GuHCl, e.g. in a concentration of 2.7to 3.9 M, and at a pH of 6.6 to 8.0. The solution is then subsequentlyheated at temperature of 55 to 61° C. for about 30 minutes to about 4hours in order to inactivate viruses, which may still be present in thesolution.

For both options, Option I and Option II, the GuHCl concentration of thesolution with a pH of 6.6 to 8.0 preferably is within the range from 3.0to 3.9 M and is most preferably 3.5 M. Within this concentration range,aggregate formation of the proteins in the solution is suppressed.

In an embodiment the pH of the solution with a pH of 6.6 to 8.0 iswithin the range from 7.0 to 8.0. In particular embodiments the pH ofthe solution is at or about 7.3. This means that the method according tothe present invention is performed at physiological (about 7.35-7.45) ornearly physiological pH, which reduces the risk of the target proteinsdenaturizing and losing their biological activity.

In the case of Option I, where the heat treatment step is performedprior to the virus filtration, the solution with a pH of 6.6 to 8.0 ispreferably prepared by suspending Apo A-I precipitate in a 4.0 to 4.6 MGuHCl and then subsequently adjusting the GuHCl concentration within therange from 2.7 to 3.9 M and the pH within the range from 6.6 to 8.0,e.g. by dilution. The GuHCl concentration may in particular be withinthe range from 2.7 to 3.5 M.

After the heat treatment, the solution for step (a) is prepared from thesolution with a pH within the range from 6.6 to 8.0 by adjusting the ApoA-I protein concentration and the GuHCl concentration to the desiredrange of the solution (A), e.g. by diluting the solution with WFI.

In the case of Option II, where the heat treatment step is performedafter the virus filtration, the solution with a pH of 6.6 to 8.0 ispreferably prepared from the solution after step b) by adjusting the ApoA-I protein concentration of the solution within the range from 5 to 30g/L and the GuHCl concentration of the solution within the range from1.3 to 3.2 M.

The specific combination of the solution comprising GuHCl and having apH within the range from 6.6 to 8.0, makes viral clearance much fasterthan according to the previously known pasteurization procedures, namelywithin about 30 minutes to about 4 hours at 60° C. instead of thewell-established 10 hours methods.

Due to the reduced time needed for achieving viral clearance, the methodof the present invention is particularly well suited for use inindustrial applications, where time is a key factor.

Furthermore, due to the shortened time that the target protein isexposed to elevated temperatures, the risk that the protein isirreversibly denatured is greatly lowered even without the addition ofstabilizers and therefore without risking the impairment of proteinfunction. This makes the method of the present invention particularlysuitable for viral removal from protein intended for therapeutic use oras a vehicle for therapeutic delivery.

Whilst the above heat treatment method is preferred it is recognisedthat the heat treatment step can also involve more traditional methods,like heating the Apo A-I solution at 60° C. for at least 10 hours.

The combination of the heat treatment and filtration steps has thepotential to enable Apo A-I to be manufactured with at least LRV 12 logLRV for parvoviruses like MVM, at least LRV 9 log for non-envelopedviruses like EMCV, and at least LRV 8.5 log for lipid enveloped viruseslike BVDV.

The present invention provides an Apo A-I preparation with at least 12log LRV (log reduction value) for a parvovirus; and/or at least 9 logLRV for a non-enveloped virus; and/or at least 8.5 log LRV for a lipidenveloped virus. In some embodiments of the present invention providesan Apo A-I preparation with at least 12 log LRV (log reduction value)for a parvovirus; and/or at least 9 log LRV for a non-enveloped virus.In some embodiments of the present invention provides an Apo A-Ipreparation with at least 12 log LRV (log reduction value) for aparvovirus. In particular embodiments the parvovirus is MVM. Inparticular embodiments the non-enveloped virus is a picomaviridae virus,such as EMCV. In particular embodiments the lipid enveloped virus is aflaviviridae virus. In particular embodiments the Apo A-I preparation issuitable for pharmaceutical use. In particular embodiments of theinvention the MVM and/or EMCV LRV for the Apo A-I preparation isdetermined by the method of Example 6.

The solutions used in the invention may further comprise additives, forexample EDTA. In particular embodiments EDTA is added in a concentrationof about 1 mM.

Whilst the methods of the present invention can be performed atlaboratory scale, they can be scalable up to industrial size withoutsignificant changes to conditions. Thus, in an embodiment disclosedherein, the methods of the present invention are performed on anindustrial or commercial scale. Preferably, the methods of the inventionare suitable for the commercial scale manufacture of humanapolipoprotein A-1 (Apo A-I). For example, when using plasma fractionsas a starting material in the method of the invention, then commercialscale manufacture would involve the use of a plasma fraction derivedfrom at least about 500 kg of plasma. More preferably, the startingplasma fraction will be derived from at least about 5,000 kg, 7,500 kg,10,000 kg and/or 15,000 kg of plasma per batch.

The purified apolipoprotein may subsequently be formulated with othercomponents to make a pharmaceutical composition, e.g. to makereconstituted HDL (rHDL). The methods of the invention may thereforecomprise an additional step of mixing the purified apolipoprotein with alipid to make an rHDL formulation. As used herein, an rHDL formulationmay be any artificially-produced apolipoprotein formulation orcomposition that is functionally similar to, analogous to, correspondsto, or mimics, high density lipoprotein (HDL) typically present in bloodplasma. rHDL formulations include within their scope “HDL mimetics” and“synthetic HDL particles”. Suitably, the rHDL formulation comprises anapolipoprotein, a lipid and, optionally, a detergent.

rHDL formulations of the invention may further comprise cholesterol. Theformulations may be produced using organic solvents, which in some casesare used for dissolving the lipid component (e.g. phosphatidylcholine)when producing the formulation, such as described in U.S. Pat. No.5,652,339. However it is preferred that the apolipoprotein formulationis produced in the absence of organic solvent.

Suitably, the apolipoprotein is at a concentration of about 5-100 g/L,preferably 10-50 g/L or more preferably 25-45 g/L This includes 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and100 g/L and any ranges between these amounts. In other embodiments, theapolipoprotein may be at a concentration of from about 5 to 20 g/L, e.g.about 8 to 12 g/L.

The lipid may be any lipid which is a component of naturally-occurringHDL or of reconstituted high density lipoprotein (rHDL). Such lipidsinclude phospholipids, cholesterol, cholesterol-esters, fatty acidsand/or triglycerides. Preferably, the lipid is a phospholipid.Non-limiting examples of phospholipids include phosphatidylcholine (PC)(lecithin), phosphatidic acid, phosphatidylethanolamine (PE) (cephalin),phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol(PI) and sphingomyelin (SM), sphingosine-1 phosphate or natural orsynthetic derivatives thereof. Natural derivatives include egg PC, eggPG, soy bean PC, hydrogenated soy bean PC, soy bean PG, brain PS,sphingolipids, brain SM, galactocerebroside, gangliosides, cerebrosides,cephalin, cardiolipin, and dicetylphosphate. Synthetic derivativesinclude dipalmitoylphosphatidylcholine (DPPC),didecanoylphosphatidylcholine (DDPC), dierucoylphosphatidylcholine(DEPC), dimyristoylphosphatidylcholine (DMPC),distearoylphosphatidylcholine (DSPC), dilaurylphosphatidylcholine(DLPC), palmitoyloleoylphosphatidylcholine (POPC),palmitoylmyristoylphosphatidylcholine (PMPC),palmitoylstearoylphosphatidylcholine (PSPC), dioleoylphosphatidylcholine(DOPC), dioleoylphosphatidylethanolamine (DOPE),dilauroylphosphatidylglycerol (DLPG), distearoylphosphatidylglycerol(DSPG), dimyristoylphosphatidylglycerol (DMPG),dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol(DSPG), dioleoylphosphatidylglycerol (DOPG),palmitoyloleoylphosphatidylglycerol (POPG), dimyristoylphosphatidic acid(DMPA), dipalmitoylphosphatidic acid (DPP A), distearoylphosphatidicacid (DSPA), dimyristoylphosphatidylethanolamine (DMPE),dipalmitoylphosphatidylethanolamine (DPPE),dimyristoytphosphatidylserine (DMPS), dipalmitoylphosphatidylserine(DPPS), distearoylphosphatidylethanolamine (DSPE),dioleoylphosphatidylethanolamine (DOPE) dioleoylphosphatidylserine(DOPS), dipalmitoylsphingomyelm (DPSM) and distearoylsphingomyelin(DSSM). The phospholipid can also be a derivative or analogue of any ofthe above phospholipids.

In some embodiments the lipid component of the rHDL comprises at leasttwo different phosphatidylcholines. In particular embodiments the atleast two phosphatidylcholines is a palmitoyl-oleoyl-phosphatidylcholine(POPC) and a di-palmitoy-lphosphatidylcholine (DPPC). In particularembodiments the reconstituted HDL formulation of the present inventioncomprises POPC and DPPC. In particular embodiments the ratio of thePOPC:DPPC is about 75:25; or about 50:50.

In other specific embodiments, the lipid is, or comprises, sphingomyelinin combination with a negatively charged phospholipid, such asphosphatidylglycerol (e.g.1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(l-glycerol)). A combinationof sphingomyelin and phosphatidylglycerol (particularly1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(l-glycerol)) is specificallyenvisaged for use as the lipid. In these embodiments, the sphingomyelinand the phosphatidylglycerol may be present in any suitable ratio, e.g.from 90:10 to 99:1 (w:w), typically 95:5 to 98:2 and most typically97:3.

Preferably the phospholipid is, or comprises, phosphatidylcholine, aloneor in combination with one or more other phospholipids. An example ofanother phospholipid is sphingomyelin. In some embodiments, theapolipoprotein formulation may comprise a detergent.

Typically, although not exclusively the lipid may be present at aconcentration of 10-100 g/L or preferably 30-60 g/L.

The detergent may be any ionic (e.g. cationic, anionic, Zwitterionic)detergent or non-ionic detergent, inclusive of bile acids and saltsthereof, suitable for use in rHDL formulations. Ionic detergents mayinclude bile acids and salts thereof, polysorbates (e.g. PS80), CHAPS,CHAPSO, cetyl trimethyl-ammonium bromide, lauroylsarcosine,n-octyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,n-decyl-N,N-dimethyl-3-ammonio-1-propane sulfonate and4′-amino-7-benzamido-taurocholic acid.

Bile acids are typically dihydroxylated or trihydroxylated steroids with24 carbons, including cholic acid, deoxycholic acid chenodeoxycholicacid or ursodeoxycholic acid. Preferably, the detergent is a bile saltsuch as a cholate, deoxycholate, chenodeoxycholate or ursodeoxycholatesalt. A particularly preferred detergent is sodium cholate.

However, high levels of detergent have been shown to be associated withliver toxicity in some systems, e.g. levels of 0.3 g/g Apo-AI or 6 g/LrHDL formulation (at 20 g/L Apo-AI). Accordingly, 5-10% of this level ofdetergent is preferred for use in the invention, i.e. 0.015-0.03 g/gApo-AI or 0.5-0.9 g/L rHDL formulation (at 30 g/L Apo-AI). The “level”of detergent may be an absolute amount of detergent, a concentration ofdetergent (e.g. mass per unit volume of rHDL formulation) and/or a ratioof the amount or concentration of detergent relative to another amountor concentration of a component of the rHDL formulation. By way ofexample only, the level of detergent may be expressed in terms of thetotal mass of apolipoprotein (e.g. Apo-AI) present in the rHDLformulation. A detergent concentration no less than about 0.45 g/L ofrHDL formulation with 30 g/L apolipoprotein is optimal in terms of bothstability and non-toxicity. Stability may advantageously be measured byany means known in the art, although turbidity of the rHDL formulationis a preferred measure. In particular embodiments the detergentconcentration is between 0.5-1.5 g/L in the rHDL formulation. Thedetergent concentration can be determined using a colorimetric assay.For example using the Gallsäuren test kit and Gallsäuren Stoppreagenswith plasma added to the reaction vials (125 μL plasma in 1 mL reactionvolume).

More generally, the level of detergent when present in the rHDLformulations of the invention is about 5-35% of that which displaysliver toxicity. This range includes, for example, 5%, 10%, 15%, 20%,25%, 30% and 35%. More preferably, the level of detergent is about 5-20%of that which displays liver toxicity. Advantageously, the level isabout 5-10% of that which displays liver toxicity. Preferably, theselevels are expressed in terms of the minimum or threshold level ofdetergent that displays liver toxicity. Liver toxicity can be evaluatedby various in vitro and in vive models. One example of an in vitro modeluses HEP-G2 cells. This involves growing HEP-G2 cells into the logphase. The cells are then removed from the culture medium and washed inPBS prior to trypsinization and resuspension in 10 mL of culture medium(90% DMEM, 10% inactivated FCS, 1% nonessential amino acids, 1%Pen/Strep). Cell growth and viability are monitored using a Neubauerhaemocytometer and trypan blue staining. Aliquots of 100 μL containing10×10⁴ C/mL are subsequently seeded, into 96 well F-bottom plates andincubated overnight at 37° C., 5% CO₂, 95% H₂O. Samples (700 μL)containing the test articles (e.g rHDL formulations) are prepared byaddition of culture medium. The medium from the first row of wells isremoved and 200 μL of the test article solution added. A serial 1:2dilution series is completed on the plates. The plates are thenincubated for 72 hours at 37′C, 5% CO₂, 95% H₂O. After which the cellviability is determined. This can be done by adding 50 μL of 3× NeutralRed Solution (70 mg Neutral Red in 100 mL PBS) to each well. The platesare incubated for 2 hours at 37° C., 5% CO₂, 95% H₂O and the wellswashed once with 200 μL PBS. After this, 100 μL of ethanol is added toeach plate and the plates shaken for 20 minutes prior to being read at540 nm. An example of an in vivo hepatoxicity model is the consciousrabbit model. The model uses rabbits which have been placed in arestraint device (rabbit holder) and i.v. catheters inserted into theirear veins. Test articles are given as a 40 minute i.v. infusion. Bloodsamples are taken from the ear artery and collected into serum andstreptokinase-plasma (5%) vials. Blood samples are processed to serum,stored at −20° C. and to plasma and stored at −80° C. Samples can thenbe assessed for the level of ALT and AST activity using enzymaticphotometric test kits available commercially (Greiner Biochemica).Whilst human Apo A-I levels can be determined using a nephelometricassay or high performance capillary electrophoresis.

In a further preferred embodiment, the rHDL formulation comprises alipid at a level that does not cause liver toxicity. Suitably, the levelof lipid is about 20-70% of that which causes, or is associated with,liver toxicity. In particular embodiments, the level of lipid ispreferably about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% of thatwhich causes, or is associated with, liver toxicity, and any rangesbetween these amounts. Preferably, these levels are expressed in termsof the minimum or threshold level of lipid that displays liver toxicity.By way of example, a level of lipid which has been shown to beassociated with liver toxicity is 84 g L. Accordingly the lipid ispreferably at a concentration of about 30-60 g/L. This includes 30, 35,40, 45, 50, 55 and 60 g/L and any ranges between these amounts. Aparticularly advantageous concentration of lipid is about 30-50 g/L, orin certain embodiments about 34 or 47 g/L. The “level” of lipid may bean absolute amount of lipid, a concentration of lipid (e.g. mass perunit volume of rHDL formulation) and/or a ratio of the amount orconcentration of lipid relative to another amount or concentration of acomponent of the fixed dosage apolipoprotein formulation. By way ofexample only, the level of lipid may be expressed in terms of a molarratio of apolipoprotein (e.g. Apo-AI) present in the fixed dosage rHDLformulation.

In one preferred embodiment, the molar ratio of apolipoprotein:lipid inthe rHDL formulations of the invention is in the range 1:20 to 1:100.This range includes molar ratios such as 1:30, 1:40, 1:50, 1:60, 1:70,1:80 and 1:90. More preferably, the molar ratio of apolipoprotein:lipidis in the range of 1:40 to 1:80; or 1:40 to 1:75; or 1:45 to 1:70; or1:40 to 1:65; or 1:40 to 1:60; or 1:40 to 1:55; or 1:40 to 1:50; or 1:45to 1:80; or 1:45 to 1:75; or 1:45 to 1:70; or 1:45 to 1:65; or 1:45 to1:60; or 1:45 to 1:55; or 1:50 to 1:80; or 1:50 to 1:75; or 1:50 to1:65; or 1:50 to 1:60. A particularly advantageous ratio ofapolipoprotein:lipid is about 1:40; or about 1:45; or about 1:50; orabout 1:55; or about 1:60.

In other embodiments, the molar ratio of apolipoprotein:lipid in therHDL formulations of the invention is in the range from about 1:80 toabout 1:120. For example, the ratio may be from 1:100 to 1:115, or from1:105 to 1:110. In these embodiments, the molar ratio may be for examplefrom 1:80 to 1:90, from 1:90 to 1:100, or from 1:100 to 1:110.

In a particular embodiment, the present invention provides an rHDLformulation obtained by the method of the invention.

In a particular embodiment, the invention provides an rHDL formulationcomprising an Apo A-I preparation with at least 12 log LRV (logreduction value) for a parvovirus; and/or at least 9 log LRV for anon-enveloped virus; and/or at least 8.5 log LRV for a lipid envelopedvirus. In some embodiments of the present invention provides an rHDLformulation comprising an Apo A-I preparation with at least 12 log LRV(log reduction value) for a parvovirus; and/or at least 9 log LRV for anon-enveloped virus. In some embodiments of the present inventionprovides an Apo A-I preparation with at least 12 log LRV (log reductionvalue) for a parvovirus. In particular embodiments the parvovirus isMVM. In particular embodiments the non-enveloped virus is apicomaviridae virus, like EMCV. In particular embodiments the lipidenveloped virus is a flaviviridae virus. In particular embodiments ofthe invention the MVM and/or EMCV LRV for the Apo A-I preparationcomprised in the rHDL formulation is determined by the method of Example6. In particular embodiments the rHDL formulation is suitable forpharmaceutical use.

The skilled person would understand that additionalpurification/fractionation steps to the two dedicated virus reductionsteps of virus filtration and heat inactivation can potentially providefurther levels of virus clearance for the Apo A-I preparations and rHDLformulations of the present invention. For example high ethanolconcentrations which are often used when obtaining Apo A-I richfractions from plasma have been shown to inactivate viruses (Hénin et.al., 1988, Vox Sang. 54(2):78-83). In such situations the LRV of thesesteps can be added to the overall LRV for the Apo A-I preparations orrHDL formulations of the present invention.

Purified Apo A-I as hereinbefore described can be formulated intopharmaceutical compositions, such as into reconstituted HDL fortherapeutic use (including as described above). Such pharmaceuticalcompositions may include a pharmaceutically acceptable carrier ordiluent. Non-limiting examples of pharmaceutically acceptable carriersor diluents include water, emulsifiers, binders, fillers, surfactants,buffers, stabilizers, salts, alcohols and polyols, detergents, proteinsand peptides, lipids, gums, sugars and other carbohydrates, althoughwithout limitation thereto.

Reconstituted HDL may, in addition to Apo-AI, comprise one or more of alipid, a detergent and a stabilizer, although without limitationthereto. Non-limiting examples of lipids include phospholipids,cholesterol, cholesterol-esters, fatty acids and/or triglycerides.Preferably, the lipid is a phospholipid. Non-limiting examples ofphospholipids include phosphatidylcholine (PC) (lecithin), phosphatidicacid, phosphatidylethanolamine (PE) (cephalin), phosphatidylglycerol(PG), phosphatidylserine (PS), phosphatidylinositol (PI) andsphingomyelin (SM) or natural or synthetic derivatives thereof.Stabilizers may be a carbohydrate such as a sugar (e.g. sucrose) or asugar alcohol (e.g. mannitol or sorbitol), although without limitationthereto. If present, the detergent may be any ionic (e.g cationic,anionic, Zwitterionic) detergent or non-ionic detergent, inclusive ofbile acids and salts thereof, such as sodium cholate.

In particular embodiments the pharmaceutical compositions are describedin WO2014/066943.

The invention also provides a method of treating or preventing adisease, disorder or condition in a mammal including the step ofadministering to the mammal Apo-A1, an Apo-A1 preparation, apharmaceutical composition or an rHDL formulation according to any ofthe aforementioned aspects to a mammal to thereby treat or prevent thedisease, disorder or condition.

Therapeutic uses for Apo A-1 and/or reconstituted HDL formulations mayinclude treatment or prophylaxis of cardiovascular disease (e.g. acutecoronary syndrome (ACS, atherosclerosis and myocardial infarction) ordiseases, disorders or conditions such as diabetes, stroke or myocardialinfarction that predispose to ACS, hypercholesterolaemia (e.g. elevatedserum cholesterol or elevated LDL cholesterol) and hypocholesterolaemiaresulting from reduced levels of high-density lipoprotein (HDL), such asis symptomatic of Tangier disease.

Certain embodiments of the invention will now be described withreference to the following examples which are intended for the purposeof illustration only and are not intended to limit the scope of thegenerality hereinbefore described.

EXAMPLES Example 1

The following example describes a sequential arrangement of the processsteps according to a preferred embodiment of the process according tothe present invention. This process includes a Fraction IV precipitatederived starting material being dissolved in the presence of GuHCl,filtered to remove filter aids prior to pH adjustment, heat treatment,dilution and virus filtration. The heat treatment can alternatively alsobe performed after the virus filtration step.

Methods and Materials Apo A-I Protein Concentration

Generally, the Apo A-I protein concentration in solution was measured bydetermining the absorbance at 280 nm using WFI (water of injection) asdiluent and is typically 5 to 30 g/L. The protein calculation is asfollows:

${{Protein}\mspace{14mu} {{concentration}\mspace{14mu}\left\lbrack {g/L} \right\rbrack}} = \frac{{Measured}\mspace{14mu} {Absorbance} \times 0.885 \times {Dilution}}{1.0844}$

Alternatively the Apo A-I protein concentration can be determined usingeither nephelometry or high performance capillary electrophoresis(Hewlett Packard 3D CE, Agilent Technology). Briefly, in relation tohigh performance capillary electrophoresis the method included thefollowing steps—samples (150 μL) containing approximately 2-3 mg/mL ApoA-I (if necessary diluted with water) were prepared with 16% SDS (25 μL)and phenylalanine (25 μL, 4 mg/mL). The samples were then incubated in awater bath for 3 minutes prior to dilution in an electrophoresis buffer(50 mM sodium borate, 0.2% SDS, 300 μL) and filtered (0.45 μm). Thesamples were then loaded onto a fused silica capillary (56 cm by 50 μmid, Agilent G1600-61232). Electrophoresis was carried out at 25 kV. Thestandard used was an International Apo A-I Standard (BCR-393). Thismethod is particularly useful when the Apo A-I is part of areconstituted HDL.

4.6 M Guanidine Hydrochloride Solution

The 4.6 M GuHCl solution with 1 mM EDTA (Titriplex) was prepared fromWFI (water for injection), GuHCl salt and EDTA. The pH of the 4.6 MGuHCl solution was adjusted to pH 7.2 to 7.4 with NaOH.

1.7 M Guanidine Hydrochloride

The 1.7 M GuHCl solution with 1 mM EDTA (Titriplex) was prepared fromWFI (water for injection), GuHCl salt and EDTA. The pH of the 1.7 MGuHCl solution was adjusted to pH 7.2 to 7.4 with NaOH.

10 mM NaCl Diafiltration Solution

The diafiltration solution was prepared from WFI and NaCl. Theconductivity of the diafiltration solution was determined at 1.0 to 1.2mS/cm.

Experimental Procedures

Solubillsation of Apo A-I Precipitate

Apo A-I precipitate was dissolved in 4.6 M guanidine hydrochloride(GuHCl), homogenized, and the pH adjusted to pH 7.3±0.1.

Clarifying Filtration

The filtration solution was clarified by depth filtration to removeresidual filter aid. The filter was prewashed with water for injection(WFI) and post-washed with 4.6 M GuHCl solution. The post-wash iscombined with the filtrate, achieving a GuHCl concentration in thefiltrate of about 3.5 M. The combined filtrate was collected and had anApo A-I concentration in the range of 0 to 30 g/L.

Before the virus filtration step, a heat treatment step was conducted toprovide virus inactivation.

Heat Treatment

For the heat treatment, the pH was adjusted to 7.1 to 7.5. The GuHClconcentration was calculated according to the following formula andadjusted to at least 3.0 M.

GuHCl[M]=−41.4−(0.0170×Protein[g/L])+(41.5×Density[g/cm3])

The mixture was then incubated at a temperature of about 60.0° C. forabout 30 minutes to about 4 hours. The mixture was returned to roomtemperature and diluted before being subjected to the virus filtrationstep.

Dilution Step

Prior to the virus filtration step, the mixture was diluted with WFI toa final GuHCl concentration of 1.5-2.0 M and an Apo A-I concentration inthe range of 0 to 30 g/L.

Virus Filtration

The purpose of the virus filtration step was to physically remove virusparticles. The prefiltration was conducted using a PALL SKL 7002 NTP 0.1μm (Nylon). Then a sterile Planova BioEx nanofilter (Asahi KaseiCorporation) with 1 m² filter area was used for the filtration step. TheBioEx filter allowed at least 340 kg of solution per m² of filtersurface area. Both pre-filter and virus-removing filter were flushed atroom temperature with 1.7 M GuHCl. The dead-end virus filtration processwas performed at room temperature. The pressure was maintained below 3.4bar. After completion of the filtration, the filter was post-flushedwith 1.7 M GuHCl. Recovery of Apo A-I across the step is excellent withvalues above 95% and typically around 100%.

Example 2

A comparison of filtration using a BioEx filter in the presence ofdifferent GuHCl (1.7 M & 3.2 M) and Apo A-I concentrations (5.8, 8.9,19.7 g/L) was conducted (FIGS. 1 to 3). The virus filtration processeswere conducted in an analogous manner to that described above in Example1.

Additional filtration studies demonstrated that lower concentrations ofGuHCl (1.0 M & 1.3 M) could result in unstable solutions that causedfilter blockage in this system. These results highlight the benefit ofoptimising GuHCl concentration levels to facilitate virus filtration.

Example 3

An Apo A-I sample was spiked with MVM at a ratio of 1:1000. The spikedsample was filtered at a protein concentration of 10 to 12 g/L through aPlanova BioEX virus removal filter in dead end mode at a pressure ofabout 2.4 bar. Samples of the filtrate were removed and assayed forresidual virus infectivity (Table 1). The results demonstrate completevirus retention was achieved across the GuHCl concentration range. Thelower GuHCl concentrations resulted in increased MVM log reductionvalues (LRV).

TABLE 1 LRV GuHCl mol/L Filtrate kg/m² Log TCID₅₀/ml ± s_(e) 2.0 460≥4.3 2.5 422 ≥4.0 3.0 502 ≥3.9 3.5 267 ≥3.6 2.0 402 ≥4.2 2.5 354 ≥4.03.0 308 ≥3.9 3.5 311 ≥3.7

In a further virus filtration study conducted under similar conditionsMVM virus breakthrough was observed in the presence of 3.4 M GuHCl.Hence concentrations below about 3.0 M GuHCl are thought to improveremoval of viruses with a diameter of approximately 20 nm (e.g.parvoviruses) using virus removal filters (such as the BioEx) in themanufacture of Apo A-I preparations.

Example 4

The study was intended to determine the effectiveness of virusfiltration in the manufacture of Apo A-I, to clear MVM from the startingmaterial. MVM was used as a model virus for very robust, smallnon-enveloped viruses including B19V.

Study Design

An Apo A-I sample was spiked with MVM at a ratio of 1000:1. The spikedsample was filtered through a Planova BioEX virus removal filter. Atdifferent time points, samples of the filtrate were removed and assayedfor residual virus infectivity.

When ≥115 g filtrate was collected, the test system was re-spiked withMVM at a ratio of 100:1. Filtration was then continued and stopped when≥8 g filtrate was collected. The filtrate was assayed for residual virusinfectivity. Finally, the filter was post-washed with at least 12 g of1.7 mol/L GuHCl solution. The post-wash fraction was collectedseparately and also analyzed for virus infectivity. In each experiment:

-   -   Cytotoxicity of the test systems was determined    -   Titer of virus stock was determined    -   Stability of the virus was determined    -   Interference was determined    -   Clearance was determined

Evaluation of Cytotoxic Effects

Cytotoxicity was assayed by a non-radioactive cell viability assay usingvirus free test system, serially diluted in cell-culture medium todetermine eventual cytotoxic effects of the sample. Samples resulting inless than 60% proliferative activity of the positive control wereconsidered cytotoxic.

Viral Interference Assay

Virus interference was performed by TCID50 end point titration utilizingthe non-cytotoxic concentration of virus free test system for the serialdilution of the stock virus. Interference was assessed by directcomparison of the virus titers obtained in interference assays to thevirus titers obtained in the standard TCID50 assays of stock virusperformed in culture medium.

Virus Titer Determination (TCID50)

Virus titers were determined by TCID50 (tissue culture infectious dose50%). Three fold serial dilutions of desalted samples were used. Viralinfectivity was assayed by endpoint titration on indicator cells in 96well plates.

Bulk Analysis (BA)

BA was used to lower the detection limit of the virus determinationassay. Viral infectivity was assayed by dispensing 9 ml of desaltedsamples each, corresponding to 3 ml of the original sample, onto two 96well plates on indicator cells.

Data Processing

Calculation of Virus Titers Determined by TCID50 on Microtiter Plates

Virus titers and their errors were calculated using the method ofSpearman & Kirber (Excel Makro: kaerber3_111.xls). Input numbers werethe results from the titer determination.

Calculation of Reduction Factors

The virus reduction factor (LRF) of the process was calculated accordingto “Virus validation studies: the design, contribution andinterpretation of studies validating the inactivation and removal ofviruses” (CPMP/BWP/268/95/Final; 14 Feb. 1996) and the requirements ofthe Bundesgesundheitsamt and the Paul-Ehrlich-Institute, Bundesamt fürSera und Impfstoffe (4. Mai 1994).

Results

Cytotoxicity

No cytotoxicity was observed in both lots of the virus-free test system.

Interference Assay

Exp. Nr. Log TCID₅₀/ml ± s_(e) 12_001.1 8.40 ± 0.12 12_001.2 8.40 ± 0.11

No interference was observed.

Bulk Analysis

Bulk analysis with 3 ml samples lowers the detection limit to ≤−0.0006log TCID50/ml (95% confidential limit). All samples tested negative forinfectious virus. Hence, complete removal of MVM was achieved throughoutthe filtrations in both experiments.

Discussion and Conclusions

The present study was intended to determine the effectiveness of virusfiltration to clear MVM from the starting material. MVM was used as amodel virus for very robust, small non-enveloped viruses and B19V.

Complete clearance of MVM by the Planova BioEX virus removal filter wasobserved, yielding a mean LRF of a ≥6.21±0.11. No cytotoxicity of thetest system and no interference with viral-infectivity were observed.

Example 5

To examine the effect of the heat treatment step on Apo A-I solutions(as described in Example 1, Heat treatment subsection) was heated to60±1° C. and spiked with MVM at a ratio of 100:1.

The spiked test system was kept at that temperature for 3 hours. Sampleswere collected at different time points and analyzed for virusinfectivity to monitor virus reduction and inactivation kineticsthroughout the process.

A sample of the test system was warmed up to 60±1° C. and kept at thistemperature throughout the whole experiment. The temperature wasmonitored and recorded during the experiment. After having reached thetarget temperature, the test system was spiked with 0.1 μm filtered MVMat a ratio of 100:1. Sample 1 was taken immediately after spiking.Additional samples were taken after 1, 5, 10, 15, 30, 60, 120 and 180minutes. All samples were diluted 1:10 with ice cold cell culture mediumand stored on ice until further processing. Then, 1 ml of the dilutedsample was passed through a PD-10 size exclusion column (GE Healthcare)in order to remove GuHCl. Briefly, 1 ml sample was applied to a column,the flow through was discarded. Then, the column was rinsed with 1 ml ofPBS, the flow through was discarded. Finally, 3 ml of PBS were appliedto the column and the desalted fraction was collected in a volume of 3ml; resulting in another 1 to 3 dilution. Then, the virus titer in thedesalted samples was determined by end point titration (TCID50).Additional samples were taken at 30, 60, 120 and 180 minutes for bulkanalysis (BA) and stored on ice until further processing. From eachsample three times 1 ml were desalted as described before. In total, 9ml of the desalted fractions were collected. The following parameterswere determined as per the methods described above in Example 4:

-   -   Cytotoxicity of the test systems    -   Titer of virus stock    -   Interference    -   Clearance

No cytotoxicity and no interference with viral infectivity were observedwith all tested samples. Rapid inactivation kinetics with completeinactivation of MVM within 30 minutes was observed. The obtained meanLRF of a ≥6.03±0.12 log indicates efficient clearance of MVM during heattreatment at 60° C. within 30 minutes.

Example 6

The studies were intended to determine the effectiveness of the twodedicated virus reduction steps—filtration and heat treatment steps toclear virus from Apo A-I solutions. The manufacturing process schemefollowed is as described in Example 1 and the spiking studies wereconducted in an analogous manner as to that described in Example 4(virus filtration) and Example 5 (heat treatment). The studiesdemonstrated the following levels of virus reduction:

Virus filtration Heat treatment Combined (LRV) (LRV) (LRV) MVM ≥6.21 ±0.11 ≥6.03 ± 0.12 ≥12.24 ± 0.23 (parvovirus) EMCV ≥4.69 ± 0.14 ≥4.75 ±0.12  ≥9.44 ± 0.26 (Picornavirus, Hepatitis A) BVDV ≥4.27 ± 0.17 ≥4.69 ±0.13 ≥8.96 ± 0.3 (Flaviviridae, Hepatitis C)

Thus the methods of the present invention enable Apo A-I to bemanufactured with at least 12 log LRV for parvoviruses like MVM, atleast 9 log LRV for non-enveloped viruses like EMCV, and at least 8.5log LRV for lipid enveloped viruses like BVDV.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications which fall within thespirit and scope. The invention also includes all of the steps,features, compositions and compounds referred to or indicated in thisspecification, individually or collectively, and any and allcombinations of any two or more of said steps or features.

1-38. (canceled)
 39. A reconstituted HDL (rHDL) formulation comprisingpurified Apo A-I, a lipid, and a pharmaceutically acceptable carrier ordiluent, wherein the purified Apo A-I is obtained by a processcomprising filtering a solution comprising Apo A-I and guanidinehydrochloride (GuHCl) through a filter having a pore size of from 15 nmto 35 nm.
 40. The rHDL formulation according to claim 39, wherein thesolution subject to filtration to obtain the purified Apo A-I comprisesApo A-I at a concentration of from 5 to 30 g/L and GuHCl at aconcentration of from 1.5 to 3.2 M.
 41. The rHDL formulation accordingto claim 39, wherein the solution subject to filtration to obtain thepurified Apo A-I has a pH from 7.1 to 7.5.
 42. The rHDL formulationaccording to claim 39, wherein the filtering to obtain the purified ApoA-I is performed at a pressure of from 0.2 to 3.4 bar.
 43. The rHDLformulation according to claim 39, wherein the filtering to obtain thepurified Apo A-I is performed at a temperature of from 18 to 26° C. 44.The rHDL, formulation according to claim 39, wherein the filtering toobtain the purified Apo A-I is performed by dead-end filtration.
 45. TherHDL formulation according to claim 39, wherein the process to obtainthe purified Apo A-I further comprises, before or after the filtering,subjecting the solution comprising Apo A-I and GuHCl to a heat treatmentfor virus inactivation.
 46. The rHDL formulation according to claim 45,wherein the solution subject to the heat treatment has a pH of from 6.6to 8.0.
 47. The rHDL formulation according to claim 45, wherein the heattreatment comprises heating the solution at a temperature of from 55 to61° C. for a period of time period of from 30 minutes±3 minutes to 4hours±24 minutes.
 48. The rHDL formulation according to claim 45,wherein the solution subject to heat treatment has a pH of from 6.6 to8.0 and comprises GuHCl at a concentration of from 2.7 M to 3.9 M. 49.The rHDL formulation according to claim 48, wherein the solution subjectto heat treatment has a pH of from 7.0 to 8.0.
 50. The rHDL formulationof claim 39, further comprising a detergent.
 51. The rHDL formulation ofclaim 50, wherein the detergent is sodium cholate.
 52. The rHDLformulation of claim 39, wherein the lipid is phosphatidylcholine. 53.The rHDL formulation of claim 39, wherein the ratio between the purifiedApo A-I and the lipid is from 1:40 to 1:75 (mol:mol), and theformulation further comprises sodium cholate at a concentration of from0.5 to 1.5 g/L.
 54. The rHDL formulation of claim 39, wherein the lipidis a mixture of sphingomyelin and phosphatidylglycerol.
 55. The rHDLformulation of claim 54, wherein the ratio between the purified Apo A-Iand the lipid is between 1:80 and 1:120 (mol:mol), and the formulationcomprises sphingomyelin and phosphatidylglycerol at a ratio of from90:10 to 99:1 (w:w).
 56. A method of administering rHDL to a mammaliansubject in need thereof, comprising administering the rHDL formulationof claim 39 to the subject.
 57. The method of claim 56, wherein thesubject is suffering from or at risk of developing one or moreconditions selected from cardiovascular disease, diabetes, stroke,myocardial infarction, elevated serum cholesterol, elevated low-densitylipoprotein (LDL) cholesterol, and reduced serum high-densitylipoprotein (HDL).